PROJECT SUMMARY/ABSTRACT Immunotherapeutic approaches which target the programmed cell death protein (PD)-1 co-inhibitory immune checkpoint have revolutionized treatment of several types of advanced cancer. However, the susceptibility of particular cell types remains highly variable and difficult to predict. We have found that both microglial cells and astrocytes govern the activity of brain-infiltrating antiviral T-cells through upregulation of PD-L1 expression in an effort to limit damaging encephalitic responses. Likewise, we have found that CD4(+) regulatory T-cells (Tregs) limit viral encephalitis through restraining expansion and activity of CD8(+) cytotoxic T-lymphocytes. While these anti-inflammatory responses within the brain are undoubtedly beneficial to the host, preventing immune-mediated damage to this vital organ, establishment of a prolonged anti-inflammatory milieu may also lead to deficiencies in viral clearance. Patients undergoing successful cART are viremically suppressed in both plasma and CSF, but persisting HIV-1 reservoirs are believed to contribute to the inability to completely cure infection. HIV-specific CD8(+) T-cell responses are critical in suppressing acute viral infection within the brain, but ultimately fail in their ability to fully eradicate virus. It is currently unknown whether glia are viable cellular targets for immune checkpoint blockade or Treg modulation. Studies proposed here are intended to fill this critical void in our understanding of how the immunosuppressive, neuroprotective brain microenvironment complicates complete clearance of viral infection. Building on our previous studies, we have developed an innovative experimental murine model in which strong CD8(+) T-cell responses specific for immunodominant HIV-1 Gag epitopes are generated within the brains of mice via heterologous prime-boost immunization with recombinant adenovirus vectors expressing the p24 capsid protein (rAd5-p24), followed by a CNS boost using Pr55Gag/Env virus-like particles (HIVLPs). This novel approach allows us to investigate the effects of immunotherapy on viral clearance from the brain in a powerful, yet convenient and inexpensive small animal model. The proposed studies will first determine whether glial cells restrain T-cell-mediated clearance of viral infection through the PD-1: PD-L1 negative immune checkpoint. Experiments proposed in Aim #2 will go on to determine whether loss or blockade of the PD-1: PD-L1 axis will facilitate anti-HIV-1 T-cell-mediated viral clearance from the brain. Finally, in Specific Aim #3 we will determine whether modulation of Treg cell activity in combination with immune checkpoint blockade will further stimulate anti-HIV-1 T-cells to promote viral clearance. The potentially synergistic combination of immune checkpoint blockade and Treg modulation is currently an area of intense investigation for treatment of a variety of immunosuppressive cancers. One of the highest priorities in contemporary HIV-1 research is to identify strategies to effect a functional cure, in which viral load is fully suppressed for extended periods in the absence of antiretroviral therapy. These studies will determine whether immunotherapy can be used to reverse immune exhaustion against the viral brain reservoir.